Method for extracting oil-soluble components from saffron stigmas and applications thereof

Abstract

The present application relates to the field of daily chemical products, and particularly relates to a method for extracting oil-soluble components from de-stalked safflower and application, comprising: subjecting the de-stalked safflower to enzymatic hydrolysis treatment by using enzyme preparation to obtain a first reactant; subjecting the first reactant to extraction and purification treatment by using ultrasonic and a eutectic solvent to obtain a second reactant, subjecting the second reactant to extraction treatment and centrifugal treatment to obtain a third reactant; and obtaining oil-soluble components from the de-stalked safflower after concentration treatment. The present application subjects the de-stalked safflower to enzymatic hydrolysis by using enzyme preparation to destroy the cell wall and promote the release of effective components, release the oil-soluble components in the cells, and improve the subsequent extraction efficiency. The reactant after enzymatic hydrolysis is purified by using ultrasonic and a eutectic solvent. The ultrasonic can strengthen the penetration and dissolution of the solvent to the target components, and the eutectic solvent can provide a more mild extraction environment, and the whole extraction method can significantly increase the extraction rate and the content of active components.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of daily chemical products, in particular to a method for extracting oil-soluble components from stigmas of saffron crocus and application thereof. BACKGROUND

[0002] Saffron crocus (Crocus sativus L), also known as saffron, is a perennial herb of the Iridaceae family. It is mainly distributed in Central Asia and Europe. As a traditional and precious medicinal material, the stigma of saffron crocus has the effects of activating blood circulation, relieving depression, and treating depression. In modern medicine, the medicinal part of saffron crocus, i.e. the stigma, has the effects of activating blood circulation, relieving depression, and treating depression. However, saffron crocus is only used for the stigma, and the yield is extremely low. Because the resources are extremely limited, it cannot meet the actual production needs, resulting in a very high price, and it has been hailed as "plant gold".

[0003] Because the stigma of saffron crocus only accounts for 7.4% of the weight of the whole flower, it means that about 170,000 flowers are picked to get 1 kg of stigmas, and the remaining stigmas are often discarded, causing great waste of resources. Therefore, the development and utilization of non-traditional medicinal parts of saffron crocus have been valued. Studies have shown that the stigmas of saffron crocus contain flavonoids, phenolic acids, monoterpenes, glycosides, alkaloids, anthraquinones, and saponins, and have antioxidant, anti-inflammatory, anti-obesity, and blood lipid abnormality activities. Therefore, the development and utilization of non-traditional medicinal parts of saffron crocus, especially the improvement of the extraction rate of oil-soluble components from stigmas of saffron crocus, have important scientific significance and application value. SUMMARY

[0004] (I) Invention purpose

[0005] The purpose of the present application is to provide a method for extracting oil-soluble components from stigmas of saffron crocus with improved extraction rate and application thereof.

[0006] (II) Technical solution

[0007] To solve the above problems, the present application provides a method for extracting oil-soluble components from stigmas of saffron crocus, comprising:

[0008] Enzymatic treatment of stigmas of saffron crocus using enzyme preparations to obtain a first reaction product;

[0009] Using ultrasonic and eutectic solvent to extract and purify the first reaction product to obtain a second reaction product, the second reaction product being a purified liquid;

[0010] Extracting and centrifuging the second reaction product to obtain a third reaction product;

[0011] Collecting supernatant of the third reactant, and obtaining oil-soluble components of the flower part of saffron crocus after concentration treatment of the supernatant.

[0012] In another aspect of the present application, preferably,

[0013] The enzyme preparation is used in an amount of 0.05-2wt% of the flower part of saffron crocus without stamens;

[0014] The enzymatic treatment temperature is 50-60℃;

[0015] The enzymatic treatment time is 60-120min;

[0016] The enzymatic treatment mode includes spraying the enzyme preparation on the flower part of saffron crocus without stamens.

[0017] In another aspect of the present application, preferably,

[0018] The enzyme preparation includes lysozyme and pectinase;

[0019] The amount of lysozyme is 1-5wt% of the enzyme preparation;

[0020] The amount of pectinase is 0.02-0.1wt% of the enzyme preparation.

[0021] In another aspect of the present application, preferably,

[0022] The deep eutectic solvent includes a hydrogen bond acceptor, a hydrogen bond donor and water;

[0023] The hydrogen bond acceptor includes betaine;

[0024] The hydrogen bond donor includes at least one of urea, xylitol, lactic acid, oxalic acid, glycerol, 1,3-propanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol and n-octanol.

[0025] In another aspect of the present application, preferably,

[0026] The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1-1:3;

[0027] The amount of water in the deep eutectic solvent is 25-45wt%;

[0028] The liquid-solid ratio of the deep eutectic solvent to the flower part of saffron crocus without stamens is 30-50:1mL / g.

[0029] In another aspect of the present application, preferably,

[0030] The power of the ultrasound is 200-600w;

[0031] The extraction time is 20-50min;

[0032] The extraction temperature is 50-70℃.

[0033] The purification treatment comprises centrifugation and membrane filtration.

[0034] In another aspect of the present application, preferably,

[0035] The extraction treatment comprises extraction using an extraction solvent.

[0036] The extraction solvent comprises at least one of caprylic / capric triglyceride, isopropyl myristate, isopropyl palmitate and isopropyl isostearate.

[0037] The volume ratio of the extraction solvent to the eutectic solvent is 1:0.5-1:1.5.

[0038] The extraction treatment time is 2-4h.

[0039] The extraction treatment is performed 2-4 times.

[0040] In another aspect of the present application, preferably,

[0041] The centrifugation treatment temperature is 25-30℃.

[0042] The centrifugation treatment centrifugal speed is 7000-9000rpm,

[0043] The centrifugation treatment time is 3-8min.

[0044] In another aspect of the present application, preferably,

[0045] The concentration treatment comprises concentration by a rotary evaporator.

[0046] In another aspect of the present application, preferably, the use of a saffron flower oil-soluble component from which the stigmas are removed in an antioxidant and anti-inflammatory medicine and cosmetic, the saffron flower oil-soluble component from which the stigmas are removed being obtained using the extraction method as described above.

[0047] (III) Beneficial effects

[0048] The above technical solution of the present application has the following beneficial technical effects:

[0049] The present application uses lysozyme and pectinase as a complex enzyme preparation to enzymatically treat the saffron flower part after removing the stigma, to destroy the cell wall and promote the release of active ingredients, release the oil-soluble components in the cells, and improve the subsequent extraction efficiency. The first reaction product after enzymolysis is purified by ultrasonic and deep eutectic solvent. Ultrasonic can enhance the penetration and dissolution of the solvent to the target components, while the deep eutectic solvent can provide a more mild extraction environment, reduce the use of traditional organic solvents, and at the same time improve the purity of the target components. The second reaction product after purification is subjected to extraction treatment to further concentrate and extract the target oil-soluble components. Subsequently, centrifugal treatment is carried out to completely separate the oil-soluble components from the components that are not soluble in oil, and the third reaction product containing the target components is obtained. Concentration treatment can further remove water and impurities, and improve the concentration and purity of the target components. The entire extraction method significantly increases the extraction rate and the content of active ingredients. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 is the overall flowchart of one embodiment of the present application;

[0051] Figure 2 is the response surface and contour plot of the extraction rate of the present application;

[0052] Figure 3 is the free radical scavenging rate plot of the oil-soluble components on DPPH and ABTS of one embodiment of the present application. DETAILED DESCRIPTION

[0053] To make the purpose, technical solutions and advantages of the present application clearer and more comprehensible, the present application will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present application. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present application.

[0054] Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application.

[0055] In the description of the present application, it should be noted that the terms "first", "second", "third" are only for descriptive purposes and should not be understood as indicating or implying relative importance.

[0056] In addition, the technical features involved in the different embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0057] EMBODIMENT

[0058] A method for extracting oil-soluble components from saffron flower parts without stigma, Figure 1 An overall flow chart of one embodiment of the present application is shown in FIG. 1, which includes the following steps: Figure 1 as shown in FIG. 1, which includes the following steps:

[0059] The enzyme preparation is used to enzymatically treat the saffron flower parts without stigma to obtain the first reactant; in this embodiment, the amount of enzyme preparation is 0.05-2wt% of the saffron flower parts without stigma; the enzymatic treatment temperature is 50-60°C; the enzymatic treatment time is 60-120min; and the enzymatic treatment mode includes spraying the enzyme preparation on the saffron flower parts without stigma. The enzyme preparation includes lysozyme and pectinase; the amount of lysozyme is 1-5wt% of the enzyme preparation; and the amount of pectinase is 0.02-0.1wt% of the enzyme preparation.

[0060] The lysozyme hydrolyzes the peptide polysaccharide-like structure in the plant cell wall, destroys the cell wall, increases its permeability, and promotes the release of more oil-soluble components in the cell wall of the saffron flower parts without stigma; at the same time, the cell wall fragments produced after the action can help to adsorb and enrich flavonoids. The pectinase specifically decomposes pectin, which exists in the plant cell wall and intercellular layer and plays a role in connecting cells, so as to make the intercellular connection loose and the tissue loose, thereby helping to release the oil-soluble components. The enzymatic treatment temperature is 50-60°C, which is conducive to the activity of the enzyme preparation. In this temperature range, the enzyme preparation can maintain high catalytic efficiency, thereby accelerating the decomposition of the flower part cell wall and releasing more oil-soluble components. The lysozyme and pectinase make the cell structure loose, opening up a path for the entry of betaine and 1,3-propanediol into the cells to extract target components, greatly improving the extraction efficiency. The enzymatic treatment time is 60-120min, which ensures that the enzyme preparation has enough time to fully act on the flower part cell wall. By adjusting the enzymatic treatment time, the extraction efficiency of the oil-soluble components can be optimized. If the time is too short, the oil-soluble components may not be fully released; if the time is too long, the activity of the enzyme preparation decreases. The enzymatic treatment mode includes spraying the enzyme preparation on the saffron flower parts without stigma, which ensures that the enzyme preparation is uniformly distributed on the surface of the flower parts, thereby improving the uniformity and efficiency of the enzymatic treatment. Through the spraying mode, the enzyme preparation can quickly penetrate into the inside of the flower part cell wall, accelerating the decomposition of the cell wall components.

[0061] The first reactant is subjected to extraction treatment by using ultrasonic and a deep eutectic solvent to obtain a second reactant, which is a liquid after purification treatment; in this embodiment, the deep eutectic solvent comprises a hydrogen bond acceptor, a hydrogen bond donor and water; the hydrogen bond acceptor comprises betaine; the hydrogen bond donor comprises at least one of urea, xylitol, lactic acid, oxalic acid, glycerol, 1,3-propanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol and n-octanol; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1-1:3; the water content in the deep eutectic solvent is 25-45 wt%; the liquid-solid ratio of the deep eutectic solvent to the flower part of the removed column head safflower is 30-50:1 mL / g. The power of the ultrasonic wave is 200-600 W; the extraction time of the extraction treatment is 20-50 min; the extraction temperature of the purification treatment is 50-70℃. The purification treatment comprises centrifugation and membrane filtration.

[0062] When ultrasonic waves propagate in a liquid, a large number of microbubbles are generated and rapidly broken, and this cavitation effect can produce a strong impact force, which helps to break the cell wall and release more oil-soluble components. The vibration of the ultrasonic wave also produces strong stirring effect, making the molecular motion in the liquid more intense, thereby accelerating the dissolution and diffusion of solutes in the solvent.

[0063] The deep eutectic solvent has unique solubility, which can dissolve a variety of organic and inorganic substances. By selecting appropriate hydrogen bond acceptors and hydrogen bond donors, the solubility of the deep eutectic solvent can be adjusted so that it can selectively dissolve oil-soluble components, thereby achieving purification. In this embodiment, betaine is used as the hydrogen bond acceptor. Betaine is a zwitterion with good solubility and stability. Urea, xylitol, lactic acid, oxalic acid, glycerol, 1,3-propanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol and n-octanol are used as hydrogen bond donors; betaine and hydrogen bond donors interact through hydrogen bonds to form a stable deep eutectic solvent system. The interaction between betaine and hydrogen bond donors reduces the melting point of the solvent, allowing it to remain in a liquid state at a lower temperature, which not only facilitates mass transfer and heat transfer during the dissolution and extraction process, but also has the advantages of low volatility, good solubility, biodegradability and environmental friendliness.

[0064] Betaine, as a hydrogen bond acceptor, can significantly improve the extraction rate of oil-soluble components from the flower parts of Crocus sativus L. due to its zwitterionic nature. The betaine-based deep eutectic solvent can also maintain the antioxidant activity of the extracted product. Betaine has good biocompatibility and low toxicity, making it suitable for use in cosmetics, food, and drug delivery systems. Betaine has excellent moisturizing properties, can absorb and retain moisture, and enhance the hydration capacity of the skin, making it an ideal moisturizer. Additionally, betaine's anti-inflammatory and antioxidant properties can alleviate skin discomfort caused by external stimuli, such as redness, itching, and allergies. Betaine can exert its effects at low concentrations, has good moisturizing and stabilizing effects, and is non-irritating to the skin and mucous membranes, making it safer for use in cosmetics and food, especially for sensitive skin and children's products. Therefore, the impact of betaine residue during the extraction process is reduced.

[0065] The amount of water used in the deep eutectic solvent is 25-45wt%. As a polar solvent, water can adjust the polarity and solubility of the deep eutectic solvent. By adjusting the amount of water, the purification effect of the deep eutectic solvent can be further optimized. The liquid-solid ratio of the deep eutectic solvent to the flower parts of Crocus sativus L. is 30-50:1 mL / g, ensuring that the deep eutectic solvent can fully contact and dissolve the oil-soluble components in the flower parts.

[0066] Under the action of ultrasound and deep eutectic solvent, oil-soluble components and part of impurity components are extracted from the flower parts of Crocus sativus L. Extraction time and temperature are key factors affecting extraction efficiency. The extraction time and temperature in this embodiment can accelerate the dissolution and diffusion of target components, thereby improving extraction efficiency.

[0067] The deep eutectic solvent composed of betaine and 1,3-propanediol combined with the changes in cell structure after enzyme treatment can more fully dissolve and extract target substances, improving the extraction rate. Betaine has the characteristics of protecting the structure and function of biological macromolecules, preventing plant components from degrading or denaturing due to environmental changes during extraction, and helping to maintain the natural structure and activity of the extract, ensuring its quality and efficacy. Compared with some traditional extraction methods, the combined treatment of betaine and 1,3-propanediol deep eutectic solvent and enzyme treatment is mild and has certain selectivity, which can more accurately extract target components, reduce the extraction of unnecessary impurities, and improve the purity of the extract. Betaine and 1,3-propanediol are relatively green and environmentally friendly substances, and the deep eutectic solvent formed is biodegradable, causing less harm to the environment during use and disposal, in line with the development trend of green chemistry. Enzymatic pretreatment makes plant cells more easily extracted, reducing the amount of deep eutectic solvent used and shortening the extraction time, thereby reducing production costs. The deep eutectic solvent of this embodiment can be recycled and reused after extraction, further improving economic efficiency.

[0068] The purification process includes centrifugation and membrane filtration steps.

[0069] The centrifugation is to separate the solid residues from the extraction liquid by centrifugation after the extraction is completed, which helps to remove impurities and unsolved solid components, and improve the purity of the extraction liquid.

[0070] The membrane filtration is to further remove the tiny particles and impurities in the extraction liquid by membrane filtration after the centrifugation is completed. It ensures that the second reactant (liquid after purification) obtained finally has high purity and transparency.

[0071] Further, in the embodiment, the hydrogen bond donor includes at least one of glycerol, 1,3-propanediol and 2,3-butanediol; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1.9; the amount of water in the deep eutectic solvent is 34wt%; the liquid-solid ratio of the deep eutectic solvent to the saffron stigmas is 40:1 mL / g, and the extraction rate of the oil-soluble components is higher. The second reactant is subjected to extraction treatment and centrifugation treatment to obtain a third reactant; the extraction treatment includes extraction using an extraction solvent; the extraction solvent includes at least one of caprylic acid / capric acid triglyceride, isopropyl myristate, isopropyl palmitate and isopropyl isostearate; the volume ratio of the extraction solvent to the deep eutectic solvent is 1:0.5-1:1.5; the extraction treatment time is 2-4h; and the extraction treatment is performed 2-4 times. The centrifugation treatment temperature is 25-30℃; the centrifugation treatment speed is 7000-9000rpm; and the centrifugation treatment time is 3-8min. Extraction is a process of transferring a compound from one solvent to another solvent by taking advantage of the difference in solubility or distribution coefficient of the compound in two mutually insoluble (or slightly soluble) solvents. In the embodiment, the extraction solvent used includes at least one of caprylic acid / capric acid triglyceride, isopropyl myristate, isopropyl palmitate and isopropyl isostearate, which have good solubility for oil-soluble components, and the volume ratio of the extraction solvent to the deep eutectic solvent is 1:0.5-1:1.5, which is to extract most of the oil-soluble components. Centrifugation is a process of separating solid particles or droplets in a mixture according to density by centrifugal force. In the embodiment, the centrifugation treatment temperature is 25-30℃, the centrifugation speed is 7000-9000rpm, and the centrifugation time is 3-8min, which are set to achieve effective separation while avoiding damage to heat-sensitive compounds.

[0072] The supernatant of the third reactant is collected, and the oil-soluble components of the Saffron Crocus flower are obtained after concentration treatment of the supernatant. The concentration treatment includes concentration by a rotary evaporator. The concentration treatment is performed by a rotary evaporator, which rotates the flask at a constant speed to increase the evaporation area, and performs heating diffusion evaporation under negative pressure to accelerate the evaporation rate, so as to separate and purify the reaction product.

[0073] The oil-soluble components of the Saffron Crocus flower are used in antioxidant and anti-inflammatory drugs and cosmetics, which are obtained by the extraction method as described above. The oil-soluble components of the Saffron Crocus flower are rich in flavonoids and have significant antioxidant and anti-inflammatory activities. Flavonoids can scavenge free radicals and inhibit oxidative stress, thereby playing a role in antioxidant drugs. In anti-inflammatory drugs, these components can reduce inflammatory reactions by inhibiting the release of inflammatory mediators. In cosmetics, the antioxidant properties of the oil-soluble components of the Saffron Crocus flower can help protect the skin from oxidative damage caused by environmental factors and delay skin aging. Its anti-inflammatory properties also help reduce skin inflammation and improve skin conditions such as redness and acne.

[0074] In this embodiment, the Saffron Crocus flower is first pretreated by enzyme method to selectively release target components, creating favorable conditions for subsequent extraction. Then, the oil-soluble components in the Saffron Crocus flower are fully dissolved and released by using betaine-based deep eutectic solvent for deep extraction, which has unique molecular structure and dissolution properties. Finally, the target oil-soluble components are precisely enriched by using oils such as caprylic acid / capric acid triglyceride for extraction. Not only does this method greatly improve the extraction rate of the oil-soluble components of the Saffron Crocus flower, but it also maximizes the biological activity of the extracted product. At the same time, this method has the outstanding advantages of green environmental protection and low cost, and uses mild reaction conditions and environmentally friendly materials throughout the process, effectively reducing the impact on the environment and production costs, and helping to further develop the Saffron Crocus related industry.

[0075] Example 1

[0076] The Saffron Crocus flower is subjected to enzymatic treatment using 0.05wt% enzyme preparation to obtain the first reactant, the petal after enzymatic treatment, the proportion of lysozyme in the enzyme preparation is 1wt%, the proportion of pectinase is 0.02wt%, the temperature of enzymatic treatment is 50℃, and the enzymatic treatment time is 60min.

[0077] The first reactant was added with a eutectic solvent, the eutectic solvent was betaine: 1,3-propanediol (the molar ratio of the two was 1:1), the water content was 25%, the liquid-solid ratio of the eutectic solvent to the saffron flower was 30:1 mL / g, the ultrasonic power was 200w, the extraction temperature was 50℃, the extraction time was 20min, and the second reactant was obtained after centrifugation and membrane filtration.

[0078] The second reactant was extracted with caprylic / capric triglyceride for 3 times, the volume ratio of caprylic / capric triglyceride to the eutectic solvent was 1:1, each extraction lasted for 3h, and the oil-soluble components were obtained after centrifugation of the combined extract, the centrifugation temperature was 25℃, the centrifugation speed was 8000rpm, and the centrifugation time was 5min. The extraction rate of the oil-soluble components was 1.98%.

[0079] Example 2

[0080] The first reactant was obtained by using 1.03wt% of enzyme preparation to enzymatically treat the saffron flower, the petal after enzymolysis, the proportion of lysozyme in the enzyme preparation was 3wt%, the proportion of pectinase was 0.06wt%, the enzymolysis treatment temperature was 55℃, and the enzymolysis time was 90min.

[0081] The first reactant was added with a eutectic solvent, the eutectic solvent was betaine: 2,3-butanediol (the molar ratio of the two was 1:2), the water content was 35%, the liquid-solid ratio of the eutectic solvent to the saffron flower was 40:1 mL / g, the ultrasonic power was 400w, the extraction temperature was 60℃, the extraction time was 35min, and the second reactant was obtained after centrifugation and membrane filtration.

[0082] The second reactant was extracted with isopropyl myristate for 3 times, the volume ratio of isopropyl myristate to the eutectic solvent was 1:1, each extraction lasted for 3h, and the oil-soluble components were obtained after centrifugation of the combined extract, the centrifugation temperature was 27.5℃, the centrifugation speed was 8000rpm, and the centrifugation time was 5min. The extraction rate of the oil-soluble components was 2.35%.

[0083] Example 3

[0084] The first reactant was obtained by using 0.2wt% of enzyme preparation to enzymatically treat the saffron flower, the petal after enzymolysis, the proportion of lysozyme in the enzyme preparation was 5wt%, the proportion of pectinase was 0.1wt%, the enzymolysis treatment temperature was 60℃, and the enzymolysis time was 120min.

[0085] The first reactant was obtained by adding a deep eutectic solvent to the safflower petals, the deep eutectic solvent being betaine: glycerol (molar ratio of 1:3), the water content of the deep eutectic solvent being 45%, the liquid-solid ratio of the deep eutectic solvent to the safflower petals being 50:1 mL / g, the ultrasonic power being 600 w, the extraction temperature being 70°C, and the extraction time being 50 min.

[0086] The second reactant was obtained by adding isopropyl isostearate to the first reactant for extraction three times, the volume ratio of isopropyl isostearate to the deep eutectic solvent being 1:1, each extraction being performed for 3 h, and then centrifuging the combined extract, the centrifugation temperature being 30°C, the centrifugation speed being 8000 rpm, and the centrifugation time being 5 min.

[0087] Examples 4-20

[0088] The first reactant was obtained by adding a deep eutectic solvent to the safflower petals, the deep eutectic solvent being betaine: glycerol (molar ratio of 1:3), the water content of the deep eutectic solvent being 45%, the liquid-solid ratio of the deep eutectic solvent to the safflower petals being 50:1 mL / g, the ultrasonic power being 600 w, the extraction temperature being 70°C, and the extraction time being 50 min.

[0089] The first reactant was obtained by adding a deep eutectic solvent to the safflower petals, the deep eutectic solvent being betaine: glycerol (molar ratio of 1:3), the water content of the deep eutectic solvent being 45%, the liquid-solid ratio of the deep eutectic solvent to the safflower petals being 50:1 mL / g, the ultrasonic power being 600 w, the extraction temperature being 70°C, and the extraction time being 50 min.

[0090] The second reactant was obtained by adding isopropyl isostearate to the first reactant for extraction three times, the volume ratio of isopropyl isostearate to the deep eutectic solvent being 1:1, each extraction being performed for 3 h, and then centrifuging the combined extract, the centrifugation temperature being 30°C, the centrifugation speed being 8000 rpm, and the centrifugation time being 5 min.

[0091] Table 1 shows the experimental parameters and results of Examples 4-20, the experimental parameters including the molar ratio of 2,3-butanediol to betaine in the deep eutectic solvent, the water content of the deep eutectic solvent, and the liquid-solid ratio of the deep eutectic solvent to the safflower petals, and the experimental parameters and results of Examples 4-20 are shown in Table 1.

[0092] Table 1 shows the experimental parameters and results of Examples 4-20, the experimental parameters including the molar ratio of 2,3-butanediol to betaine in the deep eutectic solvent, the water content of the deep eutectic solvent, and the liquid-solid ratio of the deep eutectic solvent to the safflower petals, and the experimental parameters and results of Examples 4-20 are shown in Table 1.

[0093]

[0094] Using Design-Expert software, the effects of the molar ratio of 2,3-butanediol to betaine in the eutectic solvent, the water content in the eutectic solvent, and the liquid-solid ratio of the eutectic solvent to the stigma-removed saffron petals on the extraction rate were analyzed using the regression model: Extraction rate (%) = -9.85231 + 1.224505A + 0.31635B + 0.2839C - 0.00825AB + 0.01AC + 0.00065BC - 0.449A 2 -0.00218B 2 -0.004093C 2 .

[0095] The model R 2 The value was 0.99, P < 0.01, indicating a significant model. Furthermore, the lack-of-fit term was not significant (P = 0.78 > 0.1). The lack-of-fit term is generally used to verify whether the experimental model matches the theoretical model. The results show that the model has a good fit and the experimental error is not significant. Table 2 shows the regression model and the results of the analysis of variance. The F-values ​​in Table 2 indicate the influence of each single factor on the extraction rate. The order of influence from largest to smallest is A > B > C, i.e., the molar ratio of 2,3-butanediol to betaine in the eutectic solvent > the water content in the eutectic solvent > the liquid-solid ratio of the eutectic solvent to the stigma-removed saffron flower.

[0096] Table 2 Regression Model and Analysis of Variance

[0097]

[0098] Figure 2 The response surface plot and contour plot of the extraction rate are shown, such as... Figure 2 As shown, the 3D response surface plot and contour plot can more intuitively reflect the relationship between the independent variable and the response value. The steeper the surface, the more obvious the interaction between the two factors; the closer the shape of the contour lines is to an ellipse, the more obvious the interaction between the two factors. From Figure 4, it can be seen that the influence of each single factor on the encapsulation rate is in the following order: the molar ratio of 2,3-butanediol to betaine in the eutectic solvent > the water content in the eutectic solvent > the liquid-solid ratio of the eutectic solvent to the stigmata-removed saffron petals. This is consistent with the results of the analysis of variance, proving that the regression model is reliable.

[0099] The analysis by using Design-Expert 8.0.6 software shows that the optimal preparation process is that the molar ratio of betaine: 2,3-butanediol: betaine in the eutectic solvent is 1.917:1, the water content in the eutectic solvent is 34.306%, and the liquid-solid ratio of the eutectic solvent to the flower part of Crocus sativus L. without stamens is 39.748:1. For convenience of experiment, the parameters are modified as follows: the molar ratio of 2,3-butanediol: betaine is 1:2, the water content in the eutectic solvent is 34%, and the liquid-solid ratio of the eutectic solvent to the flower part of Crocus sativus L. without stamens is 1:40. The verification shows that the extraction rate can reach 2.97%, which is consistent with the predicted value as a whole, thus indicating that the model is reliable, and the test result is ideal.

[0100] Comparative Example 1

[0101] The difference from Example 1 is only that the enzymolysis is removed, and the other steps are unchanged.

[0102] Comparative Example 2

[0103] The difference from Example 1 is only that the complex enzyme preparation is replaced by single lysozyme, and the amount is the same as the total amount of enzyme preparation in Example 1.

[0104] Comparative Example 3

[0105] The difference from Example 1 is only that the complex enzyme preparation is replaced by single cellulase, and the amount is the same as the total amount of enzyme preparation in Example 1.

[0106] Comparative Example 4

[0107] The difference from Example 1 is only that the complex enzyme preparation is replaced by cellulase and lysozyme, and the amount is the same as the total amount of enzyme preparation in Example 1.

[0108] Comparative Example 5

[0109] The difference from Example 1 is only that the eutectic solvent is not used for extraction, and only 1,3-propanediol is used for extraction, the liquid-solid ratio of the petal to 1,3-propanediol is 30:1 mL / g, the ultrasonic power is 200 w, the extraction temperature is 50°C, and the extraction time is 20 min. The other steps are unchanged.

[0110] The total flavonoid content and extraction rate of the oil-soluble components of the flower part of Crocus sativus L. without stamens prepared in Example 1 and Comparative Examples are determined according to the method in the literature “Zhang Zhihua, et al. Determination of total flavonoid content of corn oil research report [J]. Grain and oil, 2002, (09): 44.”. Table 3 shows the comparison results of Example 1 and Comparative Examples 1-3, as shown in Table 3,

[0111] Table 3 Comparison results of Example 1 and Comparative Examples 1-3

[0112]

[0113] Compared with Example 1, Comparative Examples 1, 2, 3, and 4 show that spraying a certain amount of enzyme preparation helps to extract more oil-soluble components from the stigma-free saffron flower and can significantly increase the total flavonoid content in the extraction. Furthermore, when lysozyme and pectinase are selected as enzymes, they can exert a better combined effect with eutectic solvents.

[0114] Compared with Example 1, Comparative Example 5 shows that the eutectic solvent has a better extraction effect on the oil-soluble components of saffron flowers without stigmas, and the content of total flavonoids is higher.

[0115] Example 21

[0116] Dissolve a certain amount of DPPH in anhydrous ethanol to prepare a solution with a concentration of 2×10⁻⁶. -4 A mol / L DPPH ethanol solution should be stored at 0-4℃ protected from light. A certain amount of the oil-soluble component should be prepared to a specific concentration using anhydrous ethanol.

[0117] Mix 3 mL of the test solution with 3 mL of DPPH solution and measure the absorbance at 517 nm (A1). Mix 3 mL of anhydrous ethanol with 3 mL of DPPH solution and measure the absorbance at 517 nm (A2). Mix 3 mL of anhydrous ethanol with 3 mL of the test solution and measure the absorbance at 517 nm (A3).

[0118] The DPPH free radical scavenging rate was calculated using the formula.

[0119]

[0120] Mix 5 mL of 7 mmol / L ABTS [2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt] and 88 μL of 140 mmol / L potassium persulfate, and let stand overnight at room temperature in the dark to form a free radical stock solution. Before use, dilute with anhydrous ethanol to prepare a working solution with an absorbance of 0.7 ± 0.05 at 734 nm. Take 100 μL of sample ethanol solutions of different concentrations, add 2 mL of ABTS solution, shake, and measure the absorbance A of the reaction solution at 734 nm. 测定 , with A 空白 As a positive control, absorbance was measured. The ABTS free radical scavenging rate was calculated according to the formula.

[0121]

[0122] Figure 3 The free radical scavenging rates of the oil-soluble components of one embodiment of the present invention against DPPH and ABTS are shown, such as... Figure 3As shown, the DPPH and ABTS scavenging capacity increased with the increase of sample concentration when the sample concentration was 0.1-0.5 mg / mL. When the sample concentration was 0.5 mg / mL, the DPPH radical scavenging rate and the ABTS radical scavenging rate could reach 98.95% and 97.35%, respectively.

[0123] Example 22

[0124] This example is based on the LPS (lipopolysaccharide) induced RAW264.7 macrophage inflammation model to explore the anti-inflammatory activity of the oil-soluble components of the flower part of Crocus sativus L. without stamens.

[0125] Cell culture: The high-sugar DMEM complete culture medium containing 10% fetal bovine serum (containing 100 U / mL penicillin and streptomycin) was used for culture in a 5% CO2, 37°C constant temperature incubator. The logarithmic growth period of RAW264.7 mouse macrophages was taken for experiments.

[0126] Determination of the effect of the oil-soluble components of the flower part of Crocus sativus L. without stamens on cell survival rate: the oil-soluble components were used at different concentrations (31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL) on RAW264.7 cells, and the CCK-8 kit was used to detect the effect on cell survival rate.

[0127] Determination of the effect of the oil-soluble components of the flower part of Crocus sativus L. without stamens on the inhibition of LPS-induced RAW264.7 cell release of NO, IL-8, TNF-α: the logarithmic growth period of RAW264.7 cell suspension was taken, the cell density was adjusted to 2×10 5 6 / mL, 100 μL was inoculated in each well of a 96-well plate, and the cells were adhered for 4 h, then 100 μL of two substances at concentrations of 50, 100, and 200 μg / mL and 25 μL of 1 mg / L LPS were added, respectively, and a blank group, an LPS group, and an LPS+drug group were set up, each group had 3 replicate wells, and after 24 h of culture, the Griess method was used to detect the content of NO in the supernatant, and the ELISA kit was used to detect the secretion of IL-8 and TNF-α in the supernatant.

[0128] Table 4 shows the effect of the concentration of oil-soluble components on the survival rate of RAW264.7 cells. As shown in Table 4, from Table 5, it can be seen that the oil-soluble components of the flower part of Crocus sativus L. without stamens had no toxicity to RAW264.7 cells at a concentration of 0-1000 μg / mL, i.e. the substance was within the safe concentration range when it was used on RAW264.7 cells at a concentration of 0-1000 μg / mL.

[0129] Table 4 shows the effect of the concentration of oil-soluble components on the survival rate of RAW264.7 cells. As shown in Table 4, from Table 5, it can be seen that the oil-soluble components of the flower part of Crocus sativus L. without stamens had no toxicity to RAW264.7 cells at a concentration of 0-1000 μg / mL, i.e. the substance was within the safe concentration range when it was used on RAW264.7 cells at a concentration of 0-1000 μg / mL.

[0130]

[0131] Table 5 shows the inhibitory effect of the oil-soluble components. As shown in Table 5, the oil-soluble components of the flower part of Crocus sativus L. inhibited the release of NO, IL-8 and TNF-α from LPS-induced RAW264.7 cells. Compared with the blank group, the release of NO, IL-8 and TNF-α in the cell supernatant was significantly increased after LPS stimulation (P<0.01). Compared with the LPS group, the release of NO, IL-8 and TNF-α in the cell supernatant was inhibited in each compound concentration group, and showed a concentration-dependent manner (P<0.05, P<0.01).

[0132] Table 5 shows the inhibitory effect of the oil-soluble components. As shown in Table 5, the oil-soluble components of the flower part of Crocus sativus L. inhibited the release of NO, IL-8 and TNF-α from LPS-induced RAW264.7 cells. Compared with the blank group, the release of NO, IL-8 and TNF-α in the cell supernatant was significantly increased after LPS stimulation (P<0.01). Compared with the LPS group, the release of NO, IL-8 and TNF-α in the cell supernatant was inhibited in each compound concentration group, and showed a concentration-dependent manner (P<0.05, P<0.01).

[0133]

[0134] The test results show that the anti-inflammatory effect of the oil-soluble components of the flower part of Crocus sativus L. may be related to the inhibition of the release of NO, IL-8 and TNF-α.

[0135] Example 23

[0136] An essential oil preparation with anti-inflammatory efficacy is prepared, and the formula is: caprylic / capric triglyceride 45.25%, butylated hydroxyanisole 0.05%, brassicasterol / cetylstearyl alcohol 0.5%, isostearyl isostearate 30%, bisabalol 0.2%, vitamin E acetate 0.5%, decamethylcyclopentasiloxane 20%, oil-soluble saffron 3%, macadamia seed oil 0.5%.

[0137] The preparation method is: caprylic / capric triglyceride 45.25%, butylated hydroxyanisole 0.05%, brassicasterol / cetylstearyl alcohol 0.5% are weighed in a beaker and heated to 45°C. When it is completely dissolved, it is cooled to room temperature and the remaining ingredients are added.

[0138] It should be understood that the above specific embodiments of the present application are only used for illustrative or explanatory purposes of the principles of the present application, and do not constitute a limitation of the present application. Therefore, any modification, equivalent replacement, improvement, etc. made without departing from the spirit and scope of the present application shall be included in the protection scope of the present application. In addition, the appended claims of the present application are intended to cover all variations and modifications falling within the scope and boundary of the appended claims, or the equivalent forms of such scope and boundary.

[0139] The application has been described above with the reference to embodiments. However, these embodiments are merely meant for illustrative purposes and are not meant to limit the scope of the application. The scope of the application is defined by the appended claims and their equivalents. Various substitutions and modifications could be made by those skilled in the art without departing from the scope of the application, and it is intended that all such substitutions and modifications be included within the scope of the application.

[0140] Although the embodiments of the present application have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application.

[0141] Obviously, the above-described embodiments are only examples for clearly illustrating the present application and are not intended to limit the embodiments. Based on the above description, one of ordinary skill in the art can make other different forms of changes or modifications. Here, it is not necessary and also impossible to enumerate all the embodiments. The obvious changes or modifications derived from the above are still within the protection scope of the present application.

Claims

1. A method for extracting the oil-soluble components of the saffron stigma, characterized in that, The method comprises the following steps: subjecting the de-sticked safflower flower part to enzymatic hydrolysis treatment by using an enzyme preparation to obtain a first reactant; subjecting the first reactant to extraction and purification treatment by using ultrasonic and a deep eutectic solvent to obtain a second reactant, which is a purified liquid; subjecting the second reactant to extraction and centrifugal treatment to obtain a third reactant; collecting the supernatant of the third reactant, and subjecting the supernatant to concentration treatment to obtain oil-soluble components of the de-sticked safflower flower part; wherein the amount of the enzyme preparation is 0.05-2wt% of the de-sticked safflower flower part; the enzymatic hydrolysis treatment temperature is 50-60℃; the enzymatic hydrolysis treatment time is 60-120min; the enzymatic hydrolysis treatment mode comprises spraying the de-sticked safflower flower part with the enzyme preparation; the enzyme preparation is lysozyme and pectinase; the amount of the lysozyme is 1-5wt% of the enzyme preparation; the amount of the pectinase is 0.02-0.1wt% of the enzyme preparation; the deep eutectic solvent is a hydrogen bond acceptor, a hydrogen bond donor and water; the hydrogen bond acceptor is betaine; the hydrogen bond donor is 2,3-butanediol; the molar ratio of the betaine to 2,3-butanediol is 1:2, the water content in the deep eutectic solvent is 34%, and the liquid-solid ratio of the deep eutectic solvent to the de-sticked safflower flower part is 40:1mL / g.

2. The extraction method according to claim 1, wherein the ultrasonic power is 200-600w; the extraction time is 20-50min; the extraction temperature is 50-70℃; the purification treatment comprises centrifugation and membrane filtration.

3. The extraction method according to claim 1, wherein the extraction treatment comprises extraction by using an extraction solvent; the extraction solvent is at least one of caprylic acid / capric acid triglyceride, isopropyl myristate and isopropyl isostearate; the volume ratio of the extraction solvent to the deep eutectic solvent is 1:0.5-1:1.5; the extraction treatment time is 2-4h; the extraction treatment is performed for 2-4 times.

4. The extraction method according to claim 1, wherein the centrifugal treatment temperature is 25-30℃; the centrifugal treatment rotation speed is 7000-9000rpm; the centrifugal treatment time is 3-8min.

5. The extraction method according to claim 1, wherein the concentration treatment comprises concentration by a rotary evaporator.

6. Use of an oil-soluble ingredient of a saffron stigma, which is removed from a staminate inflorescence, in an anti-inflammatory medicament, characterized in that, The oil-soluble components of the de-sticked safflower flower part are obtained by the extraction method according to any one of claims 1-5.

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